Abstract 494: Endothelial Cell Heterogeneity Governs Basal and Inducible Inflammatory Responses Relevant to Allograft Injury

Abstract

Introduction: Transplanted organs are not equally susceptible to rejection, suggesting a role for site specialization of vascular endothelial cells (EC). Most studies of leukocyte recruitment employ umbilical vein or aorta EC, and fail to capture physiological differences among EC. Methods: EC were left unstimulated or treated with TNFα or IL-1β (0.01-10ng/mL, 4-24hr). Confluent primary human EC from aorta (AEC), coronary artery (CAEC), cardiac microvessel (CMVEC), pulmonary artery (PAEC), lung microvessel (LMVEC), skin blood microvessel (DMVEC), liver sinusoids (LSEC), kidney glomerulus (RGEC) and brain microvessel (HMBVEC) were compared for expression of adhesion molecules and chemokines by Nanostring Human Immunology 594 gene panel, flow cytometry and 38-plex Luminex assay (n=3 donors). A novel adhesion assay enabled immunophenotyping of allogeneic leukocyte subsets (T, B, NK and monocytes) adherent to EC (n=4 PBMC donors). Results: >200 immunology-related genes were differentially expressed (fold >1.5, p<0.05) across untreated EC. Microvascular EC expressed higher baseline levels of ICAM-1, E-selectin and VCAM-1. TNFα and IL-1β increased these adhesion molecules, but there were clear differences in the magnitude and kinetics across cells. Most notably, LSEC failed to significantly upregulate E-selectin, and exhibited the lowest adherence of leukocytes. Only LSEC constitutively expressed suppressors of cytokine signaling (SOCS1 and SOCS3). After stimulation with TNFα and IL-1β, cardiac EC produced more fractalkine than other EC irrespective of vessel size; but large vessel (AEC, PAEC) produced the most IL-6 and G-CSF. AEC were a poor source of RANTES and IP-10, while LSEC did not increase fractalkine or GM-CSF. IL-1β increased GM-CSF, G-CSF, and GROα and IL-6. TNFα increased RANTES. More NK and B cells were recruited to TNFα than IL-1β activated EC. IL-1β selectively increased the nuclear cofactor NFKBIZ (IκBζ), but TNFα promoted Bcl-3 expression, suggesting different transcriptional programs. Conclusion: These findings demonstrate heterogeneity among EC from different vascular beds and organs. SOCS proteins may be potential therapeutic targets to reduce the inflammatory responses of ECs in multiple vascular disease models.